WNK3 kinase is a positive regulator of NKCC2 and
NCC, renal cation-Cl?cotransporters required for
normal blood pressure homeostasis
Jesse Rinehart*†, Kristopher T. Kahle*†‡, Paola de los Heros†§, Norma Vazquez§, Patricia Meade¶, Frederick H. Wilson*,
Steven C. Hebert‡, Ignacio Gimenez¶?, Gerardo Gamba§?, and Richard P. Lifton*?
Departments of *Genetics and‡Molecular and Cellular Physiology, Howard Hughes Medical Institute, Yale University School of Medicine,
New Haven, CT 06510;§Molecular Physiology Unit, Instituto de Investigaciones Biome ´dicas, Universidad Nacional Auto ´noma de Me ´xico and,
Instituto Nacional de Ciencias Me ´dicas y Nutricio ´n Salvador Zubira ´n, Tlalpan, Mexico City, 14000, Mexico; and¶Department of Pharmacology
and Physiology, School of Medicine, University of Zaragoza, 50009 Zaragoza, Spain
Contributed by Richard P. Lifton, September 26, 2005
WNK1 and WNK4 [WNK, with no lysine (K)] are serine–threonine
kinases that function as molecular switches, eliciting coordinated
effects on diverse ion transport pathways to maintain homeostasis
during physiological perturbation. Gain-of-function mutations in
either of these genes cause an inherited syndrome featuring
hypertension and hyperkalemia due to increased renal NaCl reab-
sorption and decreased K?secretion. Here, we reveal unique
biochemical and functional properties of WNK3, a related member
of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is
expressed throughout the nephron, predominantly at intercellular
junctions. Because WNK4 is a potent inhibitor of members of the
cation-cotransporter SLC12A family, we used coexpression studies
in Xenopus oocytes to investigate the effect of WNK3 on NCC and
NKCC2, related kidney-specific transporters that mediate apical
NaCl reabsorption in the thick ascending limb and distal convo-
kinase-active WNK3 is a potent activator of both NKCC2 and
NCC-mediated transport. Conversely, in its kinase-inactive state,
WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3
regulates the activity of these transporters by altering their ex-
pression at the plasma membrane. Wild-type WNK3 increases and
kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-
184 and Thr-189, sites required for the vasopressin-mediated
plasmalemmal translocation and activation of NKCC2 in vivo. The
effects of WNK3 on these transporters and their coexpression in
renal epithelia implicate WNK3 in NaCl, water, and blood pressure
homeostasis, perhaps via signaling downstream of vasopressin.
hypertension ? ion transport ? protein serine–threonine kinases
the coordinated function of diverse transcellular and paracellu-
lar electrolyte transport pathways distributed along the nephron
(1). A key step in this process is the apical entry of Na?with or
without Cl?. In the thick ascending limb of Henle (TAL) and the
distal convoluted tubule (DCT), this is mediated by related
kidney-specific electroneutral cation?Cl?cotransporters, the
Na-K-2Cl cotransporter NKCC2 (encoded by SLC12A1) in the
TAL and the Na-Cl cotransporter NCC (encoded by SLC12A3)
in the DCT. The unrelated epithelial Na?channel (ENaC;
encoded by SCNN1A, SCNN1B, and SCNN1G) mediates elec-
trogenic Na?reabsorption in the connecting tubule and collect-
reabsorption and secretion of K?and H?in these nephron
segments. Inherited variation in the activities of these flux
mediators or their regulators alters blood pressure in humans,
with increased or decreased net NaCl reabsorption raising or
lowering blood pressure (1). For example, loss-of-function mu-
tations in the genes encoding NKCC2 and NCC cause Bartter’s
and Gitelmann’s syndromes, respectively, inherited disorders
n the kidney, the regulation of net renal NaCl reabsorption is
a major determinant of blood pressure and is the product of
featuring low blood pressure due to renal NaCl wasting (2, 3).
Although a number of hormones, such as vasopressin and
aldosterone, regulate these transport proteins to maintain NaCl
and water and blood pressure homeostasis, the transducers that
link hormonal signaling to downstream targets and the mecha-
nisms coordinating the activities of multiple transporters and?or
channels are poorly understood (4).
Genetic analysis can provide fundamental insight into the
function of complex networks by identifying genes and pathways
that, when mutated, disrupt the integration of normally coor-
dinated systems (1). Recent studies have identified WNK1 and
WNK4 [WNK, with no lysine (K)] (encoded by PRKWNK1 and
PRKWNK4, respectively) as serine–threonine protein kinases
that have the biochemical properties and physiologic effects of
such integrative regulators (5). Missense mutations in WNK4
cause pseudohypoaldosteronism type II (PHAII), a disease
featuring hypertension and hyperkalemia (high serum K?levels)
due to a coupled increase in renal NaCl reabsorption and
deficiency in renal K?secretion (5). Subsequently, WNK4 has
been shown to be a multifunctional regulator of diverse Na?, K?,
and Cl?flux pathways. Wild-type WNK4 is an inhibitor of NCC,
NKCC1, the K?channel ROMK1 (or Kir1.1; encoded by
KCNJ1), and an activator of paracellular Cl?flux; some of these
effects are kinase-dependent, whereas others are independent of
WNK4’s catalytic activity (6–12). Importantly, disease-causing
missense mutations in WNK4 cluster within a highly conserved
acidic domain (5) and have sharply divergent effects on these
downstream targets; PHAII mutations eliminate inhibition of
NCC and increase paracellular Cl?permeability, whereas si-
these effects can account for the observed increase in NaCl
reabsorption and the decrease in K?secretion seen in affected
subjects. These findings demonstrate that WNK4 is a multifunc-
tional molecular switch capable of having opposing effects on
multiple ion flux pathways via independent mechanisms, pre-
cisely the sort of properties one would expect for an integrator
of systems (13). Mutations that increase expression of WNK1
cause a similar phenotype (5). Recent evidence suggests WNK1
is an upstream regulator of WNK4 at NCC (7) and may also
regulate ENaC through SGK1 (14, 15).
Conflict of interest statement: No conflicts declared.
Abbreviations: PHAII, pseudohypoaldosteronism type II; WNK, with no lysine (K); DCT,
†J.R., K.T.K., and P.d.l.H. contributed equally to this work.
?To whom correspondence may be addressed. E-mail: email@example.com, gamba@
biomedicas.unam.mx, or firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
November 15, 2005 ?
vol. 102 ?
no. 46 ?
WNK kinases are characterized by the substitution of cysteine
for lysine at a highly conserved residue in the catalytic domain;
they are found in both animal and plant species (16, 17). A total
of four such kinases exist in the human genome (5, 18), each
sharing significant homology in the kinase domain, an autoin-
hibitory domain, two putative coiled-coil domains, and a short
acidic domain. To date, little is known about the two other
members of the WNK kinase gene family, WNK2 and WNK3.
Thus, it is unknown whether these other WNK family members
act in the same pathways as WNK1 and WNK4, or whether they
have broadly different functions. If operating in the same
pathways, their functions could either be redundant to or
most abundant in brain and kidney (18, 19), but the protein has
not been localized, and its function has not been characterized.
Herein, we show WNK3’s renal localization, unique among
WNK kinases, and explore its biochemical and physiologic
functions. Our findings indicate that WNK3 has properties
distinct from those of WNK1 and WNK4 and suggest WNK3
participates in the coordinated regulation of NKCC2 and NCC,
kidney-specific cation?Cl?cotransporters necessary for electro-
lyte and blood pressure homeostasis.
cDNA Constructs. 5? EcoRI and 3? XbaI sites were engineered into
PCR primers that were used to amplify WNK3 from a full-length
human WNK3 cDNA clone (OriGene, Rockville, MD); this
amplicon was subcloned into pGH19 (9). 5? NotI and 3? EcoRI
sites and a C-terminal hemagglutinin (HA) tag were engineered
into PCR primers that were used to amplify WNK3 from the
Origene WNK3 cDNA clone, and this product was subcloned
into pCDNA3.1? (Invitrogen). QuikChange (Stratagene) was
used to introduce the D294A or Q545E mutations into pGH19-
WNK3 and pcDNA3.1-WNK3-HA. All constructs were verified
by DNA sequencing.
pSPORT1-rNCC was used for
pSPORT1-GFP-rNCC was used for quantitation of EGFP–NCC
surface expression (6). pSPORT1-rNKCC2 (20) was used for
22Na?flux studies (20).
Antibodies. Anti-WNK3 antibody was obtained from Alpha Diag-
nostics (San Antonio, TX). Other antibodies used were: anti-ZO-1
(21), anti-Megalin (gift of D. Biemesderfer, Yale University, New
anti-Tamm–Horsfall protein, anti-Calbindin (Swant, Bellinzona,
Switzerland), anti-Aquaporin-2 (Santa Cruz Biotechnology), an-
ti-HA (Santa Cruz Biotechnology), anti-rabbit IgG (Zymed), anti-
NKCC2 (T9 antibody), and anti-phospho NKCC2 (R5 antibody)
(gifts of B. Forbush, Yale University), and affinity-purified sec-
ondary antibodies conjugated to the CY2, CY3, or CY5 fluors
Transfections, SDS?PAGE, and Immunoblotting. Transfections of
WNK3 constructs into COS-7 cells were performed by using
Lipofectamine 2000 (Invitrogen). Lysates of transfected COS-7
cells or lysates from mouse or human tissues were solubilized in
sample buffer, and proteins were separated by SDS?PAGE,
transferred to nitrocellulose, blocked, and probed with anti-
WNK3 or anti-HA (each at 1:1,000 dilution), as described (6).
Membranes were then washed, incubated with secondary anti-
body, and processed with the enhanced chemiluminescence
system (Amersham Pharmacia), as described (6).
Autophosphorylation Assays. Autophosphorylation assays were
performed as described (16). WNK3-HA was expressed in
COS-7 cells, and total cell lysates were incubated with anti-HA
agarose beads (Santa Cruz Biotechnology). Beads were washed
in lysis buffer (6) and resuspended in kinase buffer (16).
Reactions were initiated by adding beads with WNK3-HA and
5 ?Ci ?32P ATP (1 Ci ? 37 GBq) (Amersham Pharmacia) in
kinase buffer. The reaction was incubated at 30°C for 30 min and
stopped by addition of sample buffer (6). Beads with WNK3-HA
were boiled in sample buffer, and phosphorylated proteins were
resolved by SDS?PAGE and visualized by autoradiography.
Immunolocalization Studies. Studies were approved by the Yale
University Animal Care and Use Committee. Mice were killed
by cervical dislocation. Excised tissues were prepared and sec-
tioned as described (21). Tissue sections were processed with
primary and secondary antibodies and visualized by immuno-
fluorescence or confocal microscopy (21). Results were consis-
tent among three different mice. Anti-WNK3 immunostaining
was competed with a 3-fold molar excess of the immunizing
peptide; staining with secondary antibody alone revealed no
Functional Assays with NCC and NKCC2.Xenopuslaevisoocyteswere
harvested and injected with cRNA of NCC or NKCC2 alone or
together with cRNA of wild-type, kinase-dead, or PHAII-like
mutant WNK3, essentially as described (8). After 4 days of
incubation, metolazone-sensitive22Na?influx (for NCC; ref. 6)
or bumetanide-sensitive86Rb?influx (for NKCC2; ref. 22) was
determined as described. NCC and NKCC2 measurements were
performed in isotonic conditions (200–210 mM). In each exper-
reproducible across at least four independent experiments for
each condition. The significance of differences between groups
of oocytes was assessed by two-tailed Student’s t test or one-way
ANOVA with multiple comparisons using Bonferroni correc-
tion, as appropriate.
Surface Expression Studies. Oocytes were injected with EGFP-
tagged NCC cRNA alone or together with wild-type or mutant
WNK3 cRNAs, incubated for 3–4 days, and membrane surface
expression of GFP-NCC was assayed by laser-scanning confocal
microscopy as described (6, 23). Total membrane fluorescence
intensity was calculated for each imaged oocyte by using SIG-
MASCAN PRO software (Jandel, San Rafael, CA; ref. 6). GFP-
NCC results are data combined from four experiments; ?12
oocytes were injected per experimental group, and each exper-
iment used oocytes from a different frog. For each injection
series, the mean fluorescence value for GFP-NCC alone was set
at 100%, and other values were expressed as percentage of this
value. The significance of differences between groups was
assessed by two-tailed Student’s t test.
NKCC2 Phospho–Protein Studies. For phospho–protein analysis,
oocytes injected with indicated constructs were incubated as
of sucrose to the medium. At the end of the incubation period,
four oocytes per group were immediately homogenized by
pipetting in 100 ?l of ice-cold antiphosphatase solution: 150 mM
NaCl?30 mM NaF?5 mM EDTA?15 mM Na2HPO4?15 mM
pyrophosphate?20 mM Hepes, pH 7.2) with 1% Triton X-100
and a protease inhibitor mixture. The homogenate was cleared
by centrifugation and supernatants collected for Western blot
previously characterized anti-NKCC2 antibody T9 and the an-
and phosphorylated NKCC2, respectively.
WNK3 Localizes to Intercellular Junctions Throughout the Nephron.
WNK3 transcripts are expressed in kidney (19). We explored the
cellular and subcellular renal localization of WNK3. Light
www.pnas.org?cgi?doi?10.1073?pnas.0508303102Rinehart et al.
immunofluorescence microscopy of kidney sections stained with
an antibody specific for WNK3 (Fig. 5, which is published as
supporting information on the PNAS web site) revealed that
WNK3, like WNK1 and WNK4, is confined to nephrons and
localizes predominantly to intercellular junctions, as demon-
strated by its colocalization with zona-occludens-1 (ZO-1), a
tight junction protein (Fig. 1). Although WNK1 and WNK4 are
confined to the distal nephron (DCT and CD) (5), WNK3 is
present in all nephron segments (Fig. 1A), with highest expres-
sion in the proximal convoluted tubule (PCT, Fig. 1B) and TAL
(Fig. 1 C–E), and lower levels of expression in the DCT and CD
(Fig. 1 F–H), as demonstrated by costaining experiments with
antibodies that serve as markers of these nephron segments (see
Methods). Confocal immunofluorescence microscopy of anti-
WNK3-stained kidney sections demonstrates that WNK3 ex-
pression extends along the lateral membrane from the level of
the tight junction to the adherens junction in all nephron
segments (Fig. 6, which is published as supporting information
on the PNAS web site).
WNK3 Is an Active Kinase. To examine WNK3’s potential kinase
activity, HA-tagged WNK3 was expressed in COS-7 cells and
immunoprecipitated from cell lysates (see Methods). Incubation
of immunoprecipitated WNK3 with ?32P-labeled ATP, followed
by electrophoresis, revealed phospholabeling of a ?200-kDa
protein, the expected size of WNK3 (Fig. 2A). The experiment
was repeated with a WNK3 mutant harboring a missense
mutation in its catalytic domain (WNK3-D294A); aspartate at
this position is highly conserved among kinases because of its
role in Mg2?binding, and alanine substitution at this site impairs
the catalytic activity of WNK1 and other kinases (16, 17).
WNK3-D294A shows virtually no autophosphorylation, indicat-
ing the dependence of phospholabeling on WNK3’s kinase
activity (Fig. 2A). In contrast, a Q545E missense mutation in
WNK3 that mimics a PHAII-causing mutation in WNK4 (5)
does not alter the phospholabeling of WNK3 (Fig. 2A). These
observations establish WNK3’s kinase activity and validate the
in subsequent experiments.
WNK3 Regulates NCC and NKCC2 by Altering Their Surface Expression.
The discrete localization of WNK3 to nephrons, along with its
localization to Cl?transporting epithelia outside the kidney (see
Kahle et al., ref. 26) and the genetic and physiologic evidence
that WNK1 and WNK4 modulate the activity of a number of
mediators of electrolyte flux in vivo, suggests specific potential
targets of WNK3 function. In particular, members of the
SLC12A family of cation?Cl?cotransporters are of interest,
because they are known to play important roles in the entry
and?or exit of Na?, K?, and Cl?in kidney epithelia, and WNK4
regulates a number of different SLC12A members (13).
Guided by these considerations, we investigated the effect of
WNK3 on NCC and NKCC2, kidney-specific SLC12A trans-
porters that mediate apical NaCl reabsorption in the TAL and
DCT, respectively, sites that express WNK3. In each case,
wild-type WNK3 and WNK3 harboring the D294A mutation
(kinase-inactive WNK3) were tested for their effects using
coexpression studies in X. laevis oocytes, a well characterized
system that has been used for the functional cloning and
physiologic characterization of these transport proteins and has
also proved useful for defining the mechanisms of their regula-
tion (20). We also tested the effect of a WNK3 mutant harboring
the Q565E mutation, which mimics a mutation that causes
PHAII in WNK4 (PHAII-like WNK3).
In contrast to WNK4’s inhibitory effect on NCC in oocytes (6,
7, 12), coexpression of WNK3 with NCC led to a dramatic
?3-fold increase in NCC activity, as measured by metolazone-
sensitive22Na?uptake (P ? 10?6, Fig. 2B). Surprisingly, kinase-
dead WNK3 not only failed to increase NCC activity but also
markedly inhibited NCC activity by ?85% (P ? 10?6; Fig. 2B).
In contrast, a PHAII-like WNK3 mutant behaved like wild-type
WNK3 on NCC (Fig. 2B). Similar effects on22Na?influx were
with EGFP-NCC (data not shown).
WNK3’s modulation of NCC activity is achieved by altering
the localization of NCC at the plasma membrane. Coexpression
of WNK3 with GFP-NCC increased GFP-NCC surface expres-
sion ?3-fold (P ? 10?6, Fig. 2C), whereas kinase-dead WNK3
Fig. 2 C and D). Thus, WNK3 is a potent regulator of NCC
activity, increasing NCC surface expression in its kinase-active
state but decreasing NCC surface expression when catalytically
Frozen mouse kidney sections were stained with anti-WNK3 antibody as
described in Methods and visualized with immunofluorescence light micros-
copy. (A) WNK3 is present in all nephron segments. Low-power view of renal
WNK3 in all nephron segments. It is apparent that WNK3 predominantly
localizes to intercellular junctions. (Original magnification, ?200.) (B) WNK3
expression in PCT. Tubule segments were stained with anti-WNK3 antibody
and identified by costaining adjacent sections with anti-Megalin antibody, a
PCT marker (not shown). (Original magnification, ?630.) (C–E) WNK3 (red)
and ZO-1 (green) immunostaining in the TAL. Tubule segments were deter-
mined by costaining experiments with anti-WNK3 and anti-Tamm–Horsfall
protein, a TAL marker (data not shown). WNK3 staining overlaps with ZO-1, a
marker of tight junctions. (Original magnification, ?630.) (F) WNK3 expres-
sion in the DCT and connecting tubule (CNT), as determined by costaining
experiments with anti-WNK3 and anti-Calbindin D-28K, a DCT?CNT marker
cortical CD (CCD), determined by costaining with anti-WNK3 (red) and anti-
WNK3 (red) and ZO-1 (green) immunostaining in the CCD. Tubule segments
were determined by costaining experiments with anti-WNK3 and anti-AQP2.
WNK3 staining overlaps with ZO-1, a marker of tight junctions. (Original
WNK3 is expressed at intercellular junctions along the nephron.
Rinehart et al.
November 15, 2005 ?
vol. 102 ?
no. 46 ?
Similarly, coexpression of WNK3 with NKCC2 in oocytes
resulted in a ?2-fold increase in NKCC2 activity, as measured
Similar to WNK3’s effect on NCC, kinase-dead WNK3 inhibited
NKCC2 activity ?70% (P ? 10?6, Fig. 2E), whereas PHAII-like
WNK3 activated NKCC2 similar to wild-type WNK3 (Fig. 2E).
Together, these data reveal that WNK3 potently increases the
activity of the mediators of apical NaCl entry in the DCT and
TAL. The magnitudes of these effects are previously unde-
scribed for any other protein kinase; moreover, WNK3 is shown
to regulate both of these transporters (20). WNK3’s activation is
specific, because its activity is reversed from activation to
inhibition by a single amino acid substitution in WNK3’s kinase
domain. WNK3’s specificity is further demonstrated by the fact
it has no effect on the activity of ENaC in Xenopus oocytes, or
on paracellular Cl?flux in Madin–Darby canine kidney II cells
(data not shown). These other renal NaCl transport processes
are regulated by WNK1 and WNK4, respectively (10, 11, 15).
86Rb?influx (P ? 10?6, Fig. 2E).
WNK3 Regulates NKCC2 Phosphorylation. To further explore the
mechanism underlying WNK3’s action on these transporters, we
focused on NKCC2, whose activation in response to vasopressin
is associated with phosphorylation of Thr-184 and Thr-189 in its
cytoplasmic N terminus (24, 25). Phosphorylation of the parala-
gous residues is necessary for and closely parallels NKCC1
activation and is also conserved in NCC (27). We monitored
phosphorylation of NKCC2 at these sites with R5, an antibody
that specifically recognizes phosphorylation of Thr-184 and
Thr-189 (24, 25). In the absence of WNK3, NKCC2 phosphor-
ylation increases from negligible levels under hypotonic condi-
mosM). In contrast, coexpression with WNK3 results in robust
NKCC2 phosphorylation under hypotonic and hypertonic con-
ditions (Fig. 3). Conversely, when kinase-dead WNK3 is ex-
pressed with NKCC2, there is a dramatic reduction in NKCC2
phosphorylation compared with the level seen with NKCC2
PHAII-like mutant WNK3 constructs were tagged with HA and expressed in COS-7 cells, purified by immunoprecipitation, and incubated with ?-32P ATP. The
products were separated by SDS?PAGE, exposed to film, and also separately stained with anti-HA antibodies. Phosphorylated WNK3 (32P-WNK3) species were
seen at 200 kDa (the size of WNK3-HA) in assays with WT and PHAII-like mutant WNK3 but were markedly reduced in assays with kinase-dead WNK3. Western
blots with anti-HA and IgG demonstrate equivalent protein loading. (B) WNK3 regulates NCC. Xenopus oocytes were injected with cRNAs encoding NCC alone
or in combination with wild-type WNK3, kinase-dead WNK3 (kin?), or PHAII-like WNK3. Metolazone-sensitive22Na?influx was measured as described in
Methods. Results are expressed as mean ? SE of metolazone-sensitive22Na?influx.*, P ? 0.0001 vs. NCC alone. WNK3 markedly increases metolazone-sensitive
22Na?influx, kinase-dead WNK3 marked inhibits this activity, whereas PHAII-WNK3 behaves like wild-type WNK3. (C and D) WNK3 regulates NCC surface
expression. (C) Oocytes were injected with cRNAs encoding EGFP-tagged NCC alone or in combination with wild-type or mutant WNK3. Surface expression of
NCC was quantitated by confocal microscopy as described in Methods. Mean fluorescence seen in oocytes expressing GFP-NCC alone is expressed as 100%; other
groups are expressed as a percentage of this value. Effects of WNK3 constructs on NCC surface expression closely parallel its effects on22Na?flux.*, P ? 10?6
vs. NCC alone. (D) Examples of confocal microscopy of oocytes expressing GFP-tagged NCC alone or with wild-type or kinase-dead WNK3. (E) WNK3 regulates
NKCC2. Oocytes were injected with cRNAs encoding NKCC2 alone or in combination with wild-type or mutant WNK3; bumetanide-sensitive86Rb?influx was
measured as described in Methods. Mean ? SE of bumetanide-sensitive86Rb?influx is shown for each group in a representative experiment. As for NCC, WNK3
increases NKCC2 activity, kinase-dead WNK3 inhibits NKCC2, activity and PHAII-like WNK3 behaves like wild-type kinase.
WNK3 kinase regulates the renal NCC and NKCC2 cotransporters. (A) WNK3 is an active kinase. Wild-type WNK3 (WT), kinase-dead WNK3 (kin?), and
www.pnas.org?cgi?doi?10.1073?pnas.0508303102 Rinehart et al.
can account for the observed inhibition of NKCC2 by kinase-
dead WNK3. These findings indicate that the effects of WNK3
on NKCC2 can be accounted for by altered phosphorylation of
regulatory sites of NKCC2.
We have shown that WNK3, a member of the WNK kinase
family, has effects that are distinct from those of WNK1 and
WNK4, demonstrating that its activity is not redundant to these
other family members. WNK3 is expressed at intercellular
DCT, in contrast to WNK1 and WNK4, which are restricted to
the aldosterone-sensitive distal nephron (5). Unlike the inhibi-
tory effect of WNK4, kinase WNK3 is a potent activator of NCC
and also NKCC2; these distinct effects suggest that either the
upstream regulators of these kinases or the timing of the effects
must be distinct to avoid a futile cycle. Kinase-inactive WNK3’s
action is reversed, strongly inhibiting NKCC2 and NCC activity.
Wild-type and kinase-inactive WNK3 regulate transporter ac-
WNK3 regulates the phosphorylation of Thr-184 and Thr-189 of
NKCC2; these sites are conserved in NCC. The phosphorylation
state of these threonines correlates with NKCC2’s activity and
plasmalemmal surface expression in vitro and in vivo (24, 25). Our
experiments suggest WNK3 regulates transporter activity by alter-
ing protein trafficking via modulation of the transporter phosphor-
ylation state. The reduced phosphorylation of NKCC2 induced by
kinase-dead WNK3 cannot be attributed to simple loss of function,
because the level of phosphorylation is lower than that seen in the
absence of WNK3. This effect of kinase-dead WNK3 could be
accounted for by direction of a phosphatase activity to the target
protein, inhibition of a kinase that normally maintains phosphor-
ylation of the target, or both. Further experiments will be needed
to clarify WNK3’s mechanism of action.
WNK3 is not only the most potent activator reported for
NKCC2 or NCC; it is also the first kinase reported to regulate
both of these transporters (20). In the kidney (Fig. 4), kinase-
active WNK3’s activities in the TAL and DCT are inferred to
promote increased renal NaCl reabsorption in nephron seg-
ments that normally mediate the reabsorption of ?30% and
?7% of the filtered NaCl load, respectively. The absence of an
effect of WNK3 on ENaC and paracellular Cl?flux, processes
that occur in the more distal CD, suggest that WNK3 activity can
shift NaCl reabsorption toward more proximal nephron seg-
ments, a key difference from WNK1 and WNK4, whose action
has been shown in part to lie downstream of aldosterone in more
distal parts of the nephron (14). Nonetheless, the absence of
effect on these latter pathways must be interpreted with some
caution, because it is possible that additional cellular compo-
nents required for a regulatory effect of WNK3 are absent from
the oocyte and Madin–Darby canine kidney cell systems used in
this study. The activity of WNK3 on NKCC2 and its presence in
the TAL, where vasopressin is known to regulate NKCC2
activity (24, 28, 29), plus the fact that WNK3 and vasopressin
induce phosphorylation of the same N-terminal threonines (24),
suggests that WNK3 might lie downstream of vasopressin sig-
naling. This may apply to NCC in the DCT as well (29). Because
the reabsorptive capacity of the TAL?DCT for NaCl via
NKCC2?NCC is large, activation of this pathway by WNK3
under conditions of intravascular volume depletion and?or high
serum osmolarity could promote increased NaCl and water
reabsorption, thereby defending intravascular volume and
plasma tonicity. The effects of the WNK3 kinase-inactivating
mutation on NKCC2 and NCC are intriguing and may mimic a
normal in vivo phenomenon, perhaps achieved by phosphoryla-
tion or altered interaction with other proteins?ligands. In its
inactive state, basal repression of NKCC2 and NCC might take
place, whereas in its active state, WNK3 could facilitate robust
NaCl and water reabsorption.
WNK3’s regulation of transporters that are expressed in other
nephron segments remains to be fully defined. In contrast to
WNK1, WNK3 does not appear to regulate ENaC, the main
mediator of NaCl reabsorption in the connecting tubule and CD.
Potential targets in the CD include the K?channel ROMK1 and
the water channel aquaporin 2. The targets of WNK3 in the PCT
are also unknown. KCC3 and KCC4, K-Cl cotransporters in the
SLC12A family highly expressed in the PCT (20), are prime
candidates, as is the Cl??formate exchanger CFEX (9). Fur-
thermore, the functional significance of WNK3 at the tight
junction is unclear; whereas WNK4 facilitates paracellular Cl?
flux in Madin–Darby canine kidney II cells (10, 11), WNK3 has
were injected with the indicated constructs and incubated at varying extra-
cellular osmolarities as indicated. After incubation oocytes were lysed and
Western blotting was performed by using the R5 (anti-phospho-NKCC2) or T9
(anti-NKCC2) antibodies as described in Methods. Phosphorylation of NKCC2
normally increases from negligible levels under hypotonic conditions (200
mM) to complete phosphorylation under hypertonic conditions (380 mM). In
contrast, coexpression of NKCC2 with kinase-active WNK3 results in robust
phosphorylation of NKCC2 at all osmolarities. Expression of kinase-dead
WNK3 results in marked reduction of NKCC2 phosphorylation under hyper-
WNK3 modulates the phosphorylation of NKCC2. Xenopus oocytes
in the TAL and DCT of the nephron, WNK3 could modulate NaCl and water
reabsorption and therefore blood pressure. Kinase-active WNK3 might in-
crease NaCl reabsorption, whereas kinase-inactive WNK3 (WNK3 kin?) might
inhibit NaCl reabsorption. These active?inactive states of WNK3 may be
achieved dynamically by ligands (e.g., downstream of vasopressin) binding to
or dissociating from the kinase.
Rinehart et al.
November 15, 2005 ?
vol. 102 ?
no. 46 ?
no effect in the same model system. These issues require future
Because individual WNK kinases have different expression
profiles, unique target specificities, and opposing effects at
common targets, it is tempting to speculate that different WNKs
may be recruited individually or in combination, enabling mod-
ulation of the activities of their targets over a wide range in
response to physiological stimuli. For example, it would be
logical if WNK4 signaling was downstream of angiotensin II
signaling and WNK3 was downstream of vasopressin signaling.
Mutations in humans in WNK1 and WNK4 have large effects to
alter the balance between NaCl reabsorption and K?secretion,
demonstrating their physiologic relevance. Our findings suggest
that WNK3 is integral to the regulation of NKCC2 and NCC,
proteins necessary for normal blood pressure homeostasis.
These observations add to the growing recognition that WNK
family members play diverse and important roles in integrated
We thank Gerhard Giebisch, Cecilia Canessa, and Gordon MacGregor
for advice and helpful discussions. This work was supported in part by
grants from the National Institutes of Health (Specialized Center of
Research Grant in Hypertension to R.P.L., DK-36803 to S.C.H. and
G.G., and DK-64635 to G.G.) and the Wellcome Trust (GR070159MA
to G.G.). K.T.K. is a trainee of the National Institute of Health Medical
Scientist Training Program. I.G. is a Ramo ´n y Cajal Investigator of the
Spanish Ministry of Education and Science. R.P.L. is an Investigator of
the Howard Hughes Medical Institute.
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